Interferon (IFN)-gamma is essential but not sufficient to control leishmaniasis. It is known that IFN-gamma is one of the major macrophage-activating cytokines, and the activated macrophages are a principal source of interleukin (IL)-12, which induces autocrine macrophage activation. In this study, the combined effect of IFN-gamma and IL-12 on the susceptibility of macrophages to Leishmania major infection was evaluated. Macrophages pretreated with IFN-gamma and/or IL-12 were infected with the parasites. Four hr post-infection (p.i.), the levels of infection and parasite load in the macrophages treated with the combination of IFN-gamma and IL-12 (IFN-gamma/IL-12) were significantly lower than those in the nontreated cells. However, the macrophages treated with either IFN-gamma or IL-12 did not show resistance to L. major infection. In addition, 72 hr p.i., the IFN-gamma/IL-12-treated and IFN-gamma-treated macrophages showed significantly lower levels of infection and parasite load than the nontreated cells, and higher levels of resistance was observed in the IFN-gamma/IL-12-treated macrophages than in the IFN-gamma-treated macrophages. Although IFN-gamma/IL-12 treatment of macrophages prior to the infection led to the induction of resistance, as described above, this resistance was not induced when these cytokines and the parasites were added simultaneously to the macrophage culture. These results suggest that IFN-gamma/IL-12 treatment prior to the infection restricts the early phase of the infection.